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- PDB-6ayg: Human Apo-TRPML3 channel at pH 4.8 -

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Basic information

Entry
Database: PDB / ID: 6ayg
TitleHuman Apo-TRPML3 channel at pH 4.8
ComponentsMucolipin-3
KeywordsTRANSPORT PROTEIN / ion channel / TRP channel / lysosomal
Function / homology
Function and homology information


NAADP-sensitive calcium-release channel activity / inner ear auditory receptor cell differentiation / monoatomic anion channel activity / TRP channels / sodium channel activity / autophagosome membrane / potassium channel activity / locomotory behavior / calcium ion transmembrane transport / calcium channel activity ...NAADP-sensitive calcium-release channel activity / inner ear auditory receptor cell differentiation / monoatomic anion channel activity / TRP channels / sodium channel activity / autophagosome membrane / potassium channel activity / locomotory behavior / calcium ion transmembrane transport / calcium channel activity / late endosome membrane / early endosome membrane / lysosomal membrane / lipid binding / plasma membrane
Similarity search - Function
: / Mucolipin / : / Mucolipin, extracytosolic domain / Polycystin cation channel, PKD1/PKD2 / Polycystin cation channel
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.65 Å
AuthorsZhou, X. / Li, M. / Su, D. / Jia, Q. / Li, H. / Li, X. / Yang, J.
Funding support China, United States, 9items
OrganizationGrant numberCountry
National Basic Research Program of China (973 Program)2014CB910301 China
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM085234 United States
National Natural Science Foundation of China (NSFC)31370821 China
Top Talents Program of Yunnan Province2011HA012 China
High-level Overseas Talents of Yunnan Province China
China Youth 1000-Talent Program of the State Council of China China
Beijing Advanced Innovation Center for Structural Biology China
Tsinghua-Peking Joint Center for Life Sciences China
National Natural Science Foundation of China (NSFC)31570730 China
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Cryo-EM structures of the human endolysosomal TRPML3 channel in three distinct states.
Authors: Xiaoyuan Zhou / Minghui Li / Deyuan Su / Qi Jia / Huan Li / Xueming Li / Jian Yang /
Abstract: TRPML3 channels are mainly localized to endolysosomes and play a critical role in the endocytic pathway. Their dysfunction causes deafness and pigmentation defects in mice. TRPML3 activity is ...TRPML3 channels are mainly localized to endolysosomes and play a critical role in the endocytic pathway. Their dysfunction causes deafness and pigmentation defects in mice. TRPML3 activity is inhibited by low endolysosomal pH. Here we present cryo-electron microscopy (cryo-EM) structures of human TRPML3 in the closed, agonist-activated, and low-pH-inhibited states, with resolutions of 4.06, 3.62, and 4.65 Å, respectively. The agonist ML-SA1 lodges between S5 and S6 and opens an S6 gate. A polycystin-mucolipin domain (PMD) forms a luminal cap. S1 extends into this cap, forming a 'gating rod' that connects directly to a luminal pore loop, which undergoes dramatic conformational changes in response to low pH. S2 extends intracellularly and interacts with several intracellular regions to form a 'gating knob'. These unique structural features, combined with the results of electrophysiological studies, indicate a new mechanism by which luminal pH and other physiological modulators such as PIP regulate TRPML3 by changing S1 and S2 conformations.
History
DepositionSep 8, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 8, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 22, 2017Group: Database references / Category: citation
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 6, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 20, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Mucolipin-3
B: Mucolipin-3
C: Mucolipin-3
D: Mucolipin-3


Theoretical massNumber of molelcules
Total (without water)258,5034
Polymers258,5034
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16780 Å2
ΔGint-162 kcal/mol
Surface area96880 Å2
MethodPISA

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Components

#1: Protein
Mucolipin-3 / Transient receptor potential channel mucolipin 3 / TRPML3


Mass: 64625.785 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MCOLN3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q8TDD5

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: human TRPML3 channel at pH 4.8 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 4.8
Details: TRPML3 was purified in HEPES buffer pH 7.4. Sodium acetate pH 4.6 was added before freezing, which made the pH to be 4.8
Buffer component
IDConc.NameFormulaBuffer-ID
118 mMHEPESC8H18N2O4S1
2135 mMsodium chlorideNaCl1
30.45 mMLauryl Maltose Neopentyl GlycolC47H88O221
4100 mMsodium acetateCH3COONa1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil holey carbon grid R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansWidth: 7676 / Height: 7420 / Movie frames/image: 32 / Used frames/image: 1-32

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Processing

EM software
IDNameVersionCategoryDetails
1RELION1.4particle selectionto automatically pick particles
2EMAN2particle selectionto semi-automatically pick particles
3Etas1image acquisition
5CTFFIND3CTF correction
11RELION1.4initial Euler assignment
12RELION1.4final Euler assignment
13RELION1.4classification
14RELION1.43D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42559 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL

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